Study On Cutting Propagation And Tissue Culture Techniques Of Michelia ’Zhongshanhanxiao’

Michelia’Zhongshanhanxiao’is a superior offspring naturally produced by the pollination of Michelia compressa.It has characteristics such as large leaves,fragrant flowers,rapid growth,and excellent cold resistance.Currently,there is a shortage of Michelia’Zhongshanhanxiao’seedlings,which cannot meet the market demand.It needs to be rapidly propagated through asexual reproduction methods,but there have been no reports on asexual reproduction techniques for Michelia’Zhongshanhanxiao’.In this study,the cutting propagation technology of Michelia’Zhongshanhanxiao’was studied,and the formation mechanism of adventitious root was discussed according to the changes of nutrients,oxidase,endogenous hormones and other contents in the process of cutting rooting,combined with transcriptome sequencing and bioinformatics analysis.At the same time,using top buds and lateral buds with stem segments as explants,the seedling tissue culture techniques of Michelia’Zhongshanhanxiao’were studied from the aspects of explant disinfection,bud induction medium,bud proliferation medium,and callus induction medium.The research results have theoretical significance and application value for the asexual reproduction of Michelia’Zhongshanhanxiao’seedlings,and also provide a reference for further research on the tissue culture technology of Michelia’Zhongshanhanxiao’.The main results are as follows:（1）Michelia’Zhongshanhanxiao’cuttings produced callus 20 days after cutting and adventitious root 40 days later.Both callus and skin can produce adventitious roots,belonging to the mixed rooting type,but most of them are callus rooting types.The skin rooting type was characterized by high position adventitious roots.（2）The propagation substrate has a significant impact on the rooting of Michelia’Zhongshanhanxiao’cuttings.When the substrate is peat soil,it exhibits the best rooting effect.After 80 days of cutting,the rooting rate was 55.56%,the number of roots was 2.06,the root length was 9.17 cm,the longest root length was 9.89 cm,and the root effect index was 0.63.（3）The application of plant growth regulators significantly affects the rooting of Michelia’Zhongshanhanxiao’cuttings.The treatment of 2000 mg·L^-1NAA for 3 minutes is the best combination of rooting hormones.After 80 days of cutting,the rooting rate was 56.67%,the number of roots was 3.67,the root length was 9.86 cm,the longest root length was 11.50 cm,and the root effect index was 1.21.（4）During the rooting process of Michelia’Zhongshanhanxiao’cuttings,there were significant differences in the content of nutrients,oxidase,and endogenous hormones in the cuttings among different treatments.Before the formation of adventitious root,the content of soluble sugar in cuttings decreased first and then increased,while the content of soluble protein changed on the contrary;NAA treatment can promote the activities of POD and PPO within cuttings,while inhibiting the activity of IAAO.The activities of the three enzymes show a trend of first increasing and then decreasing;NAA treatment promoted the content of IAA and GA₃in cuttings,while inhibited the content of ABA and ZR.After cutting,the content of IAA,ABA and GA₃reached a valley on the 20th day,while the content of ZR increased after reaching a valley on the 40th day.（5）Gene expression changed significantly in different stages of adventitious root development.GO functional enrichment analysis showed that 5 GO entries,including protein metabolism and nucleotide binding,were significantly enriched only during the callus formation stage;Hormone regulation and cytokine metabolism were significantly enriched during the formation and elongation of adventitious root.The enrichment analysis of KEGG pathway showed that genes involved in starch and sucrose metabolism,phenylpropane biosynthesis and plant hormone signal transduction played an important role in adventitious root development.Combined with GO function and KEGG pathway enrichment analysis,it was found that SAUR,ARF and ABF gene family were involved in plant hormone signal transduction pathways and promoted the development of adventitious root of cuttings.（6）In the tissue culture of Michelia’Zhongshanhanxiao’seedlings,the best disinfection method was 75%alcohol for 30 s,and then 5%Na Cl O for 12 min;The best disinfection method for lateral buds with stem segments was to soak them in 75%alcohol for 30 seconds,and then disinfect them with 5%Na Cl O for 20 minutes.The optimal induction medium for both adventitious buds was improved WPM medium+0.05 mg·L^-16-BA+0.5 mg·L^-1NAA+0.5mg·L^-1IBA,The optimal proliferation medium was improved WPM medium+0.5 mg·L^-1KT+0.8 mg·L^-16-BA+0.15 mg·L^-1IBA,The best callus induction medium was improved WPM medium+3.0 mg·L^-16-BA+0.25 mg·L^-1IBA.